Regulatory CD25+CD4 T cells are important mediators of immunologic tolerance and are likely to become a therapeutic target. However, their mechanisms in vivo remain largely unknown, and are the main focus of this proposal. The summary of the preliminary work described here shows that 1) we are able to purify in numbers sufficient for the proposed experiments antigen-specific regulatory CD25+CD4 T_;_.!!._ from wild-type TCR Tg mice and antigen-specific regulatory CD25+CD4 T cells expressing a single known TCR; 2) we have established two complementary in vivo systems to measure suppressive effects of CD25+CD4 T cells on responder T cells in non-lymphopenic animals; 3) we are able to simultaneously visualize the behavior and cellular interactions of antigen-specific CD25+CD4 T cells, antigen-specific responder T cells, and antigen-presenting dendritic cells; 4) CD25+CD4 T cells reduce the size of the T cell population responding to antigen and inhibit production of effector Thl and Th2 cytokines; and 5) CD25+CD4 T cells inhibit homeostatic proliferation of naive T cells in lymphopenic mice. The laboratory is now exceptionally poised to pursue the two specific aims in the proposal. In the first aim we will define the cellular mechanisms of by which CD25+CD4 T cells inhibit the antigen-specific T cell responses within the secondary lymphoid tissues. We will determine the fates of the dendritic cells presenting the antigen as well the antigen-specific CD25+CD4 T cells and responder T cells. In the second aim we will determine how CD25+CD4 T cells shape the development of the T cell compartment during immune reconstitution by homeostatic proliferation. In particular, we will test how CD25+CD4 T cells may regulate potentially auto-aggressive T cell clones under conditions of lymphopenia. Although cellular mechanisms constitute the main focus of this proposal, the experimental systems described are robust and are ideally suited for testing roles of specific molecules in CD25+CD4 T cell-mediated immune regulation.